PCR (Polymerase Chain Reaction) detection primer and detection method for borrelia burgdorferi

A technology for Borrelia burgdorferi and detection methods, applied in biochemical equipment and methods, methods based on microorganisms, measurement/inspection of microorganisms, etc., can solve problems such as food safety threats, achieve short detection cycle, high specificity, Highly stable effect

Inactive Publication Date: 2017-01-04
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a threat to people's food safety, so far there is no rapid detection method for this pathogen, and a rapid detection method for Borrelia burgdorferi is urgently needed

Method used

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  • PCR (Polymerase Chain Reaction) detection primer and detection method for borrelia burgdorferi
  • PCR (Polymerase Chain Reaction) detection primer and detection method for borrelia burgdorferi

Examples

Experimental program
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Effect test

Embodiment 1

[0025] The PCR detection of Borrelia burgdorferi in embodiment 1 sick poultry

[0026] After the poultry whole blood was collected and centrifuged at 1000r / min for 10min, take 200-500μL of the light white suspension at the junction of serum and blood cells and put it into a 1.5mL centrifuge tube. The collected matter in the centrifuge tube was centrifuged at 12000r / min for 10min, then the supernatant was discarded, and the precipitate was collected for nucleic acid extraction; at the same time, the standard strain culture of Borrelia burgdorferi was set up as a positive control; SPF chicken blood was used as a negative control.

[0027] Nucleic acid extraction: Add 500 μL of extraction buffer (100 mmol / L NaCl, 10 mmol / L Tris-Cl pH 8.0, 25 mmol / L EDTA pH 8.0, final concentration 10 g / L SDS and After suspending proteinase K at a final concentration of 0.1 g / L, react in a water bath at 55 °C for 2 h, and set up a culture of a standard strain of Borrelia burgdorferi as a positive ...

Embodiment 2

[0039] The specificity test of the PCR detection method of embodiment 2 Borrelia burgdorferi

[0040] Chlamydia, Mycoplasma and Theileria three pathogens with similar symptoms after infecting poultry were used as the test strains for the specificity experiment.

[0041] For nucleic acid extraction, nucleic acids were extracted from the pure cultures of the three tested pathogens as templates for PCR amplification. Obtain nucleic acid solution for use.

[0042] Configuration and amplification parameters of the PCR amplification system:

[0043] Amplification system: Each 25 μL amplification system contains 12.5 μL of 2×PCR Premix buffer, 0.5 μL each of 25 μmol / L SEQ ID NO.1 and SEQ ID NO.2, 1 μL of nucleic acid solution obtained in the nucleic acid extraction step, ultrapure water Make up the volume to 25μL;

[0044] Amplification parameters: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles ...

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Abstract

The invention belongs to the technical field of biological detection and in particular relates to a PCR (Polymerase Chain Reaction) detection primer and a detection method for borrelia burgdorferi. By extracting borrelia burgdorferi nucleic acid, a pair of borrelia burgdorferi specific primers SEQ ID NO.1 and SEQ ID NO.2 are subjected to PCR amplification; an amplified product is subjected to electrophoresis on lipolysaccharide gel through utilizing a TAE buffering solution; a PCR product is observed under an ultraviolet projector and is compared with a standard molecular weight, so as to judge whether a sample carries a borrelia burgdorferi pathogen or not: if an electrophoretogram has a 225bp specific strip, the electrophoretogram shows that the sample carries the borrelia burgdorferi pathogen; if the electrophoretogram has no 225bp specific strip, the electrophoretogram shows that the sample does not carry the borrelia burgdorferi pathogen. Detection verifies that a detection system provided by the invention has the characteristics of high specificity and good stability.

Description

technical field [0001] The invention belongs to the technical field of biological detection and relates to a PCR method for detecting Borrelia burgdorferi pathogenic nucleic acid. Background technique [0002] Borrelia burgdorferi belongs to Kingdom Monera, Spirochaetidae, Spirochaetaceae, Borrelia (also known as Borrelia). Gram staining is negative, but it is not easy to stain. Giemsa staining and fluorescent dye staining are good, and elongated spirochetes can be seen under the microscope. Borrelia burgdorferi has multiple flagella, 3-10 large and sparse helices, 10-30 μm long and 0.2-0.25 μm wide, and consists of four parts: surface layer, outer membrane, flagella and protoplasm. In liquid medium, several spirochetes can be wound together irregularly and move in a twisted state. Transmitted by ticks, it can infect birds and other animals and cause avian spirochetosis (Avian Spirochaetosis, AS). There is a threat to people's food safety, so far there is no rapid detecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689Y02A50/30
Inventor 刘生峰杨俊陆丽华王国民李贤良张雷聂福平王昱
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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